The presence of Msh2-Msh3 can significantly influence the activity of both Rad27 and Cdc9 on their cognate substrates; therefore, we asked whether it could affect their combined activity on TNR-containing replication intermediates. During replication at TNR tracts, the polymerase has the potential to pause and dissociate from the DNA (Kang et al., 1995
; Viguera et al., 2001
). Such events allow for the newly synthesized strand and displaced flap to compete for annealing to the template (Liu and Bambara, 2003
). To simulate such intermediates, we designed equilibrating flap substrates in which the downstream and upstream primers overlap and compete for template annealing over a 30 nt region. Therefore, the substrate can equilibrate into different conformations, with either 5’ and/or 3’ flaps of various sizes, depending on how the primers anneal to the template. The equilibrating sequences consisted of either ten CTG repeats or 30 nt of a randomly generated sequence, which was predicted not to form any structure (). Similar to previous reports showing that such large flaps cannot be effectively ligated (Veeraraghavan et al., 2003
), we did not observe the formation of a fully expanded product (104 bp) in the presence of Cdc9 (, lane 3). Incubation of the substrate with Rad27 and Cdc9 resulted in the majority of products being correctly processed (74 bp) (, lane 4), since Rad27 effectively cleaved at the base of the flap, creating a nick for Cdc9 to seal. Interestingly, a minority of various length expansion products was observed only with the substrate containing CTG repeats (, lane 4) and not the randomly generated sequence (, lane 12). Significantly, the titration of Msh2-Msh3 into reactions containing both Rad27 and Cdc9 resulted in the decrease of the correctly processed product and the preferential increase of only smaller expansion products for TNR substrates, corresponding to one or two repeats (, lanes 5–8, ). This increased the ratio of partially expanded to correctly processed products ~ 4.3 fold as compared to the ratio produced without Msh2-Msh3.
Msh2-Msh3 interferes with Rad27 processing and Cdc9 efficiency to create expanded sequences
It was previously reported that Rad27 cleavage of such intermediates containing TNRs generates multiple cleavage products because the two competing sequences interact with the template at various positions and equilibrate into different conformations (Liu et al., 2009
; Veeraraghavan et al., 2003
). This produces various length 5’ flaps that can be cleaved and ligated to the upstream primer to create expanded products (). In order to determine whether these expanded products could also be a result of Rad27 cleavage upstream of the flap base, we examined substrates containing only a static 5’ flap consisting of either ten CTG, four CTG, or four CAG repeats. Alternate Rad27 cleavage patterns could be observed (Fig S3a
), presumably responding to secondary structure formation within the 5’ flap (Vallur and Maizels, 2010
). Even though Rad27 cleaved at multiple sites upstream of the base (Fig S3a
), only a single 2 nt expansion product was observed with the addition of Cdc9 (Fig S3b and S3c
), corresponding to a single flap conformation that can be processed into an expanded sequence for this static substrate. Interestingly, the presence of Msh2-Msh3 in the reactions promoted the accumulation of the expanded product and decreased the formation of the correctly processed product (Fig S3b and S3c
). These results indicate that cleavage and ligation patterns of TNR-containing 5’ flaps can be altered by the formation of structure within the flap, an effect that is enhanced by the interaction with Msh2-Msh3.
Our findings with a large equilibrating flap indicate that Msh2-Msh3 favors the creation of small expansions instead of large. We, therefore, investigated the effect of Msh2-Msh3 on a smaller equilibrating flap substrate that can efficiently ligate into an expanded sequence. This substrate was designed to contain small equilibrating 9 nt flaps consisting of three CTG repeats, that would compete for annealing to the template. Additionally, the downstream primer contained seven more CTG repeats that could fully anneal to the template without competition, in order to enhance the possible structural conformations the substrate could adopt. Incubation with Rad27 resulted in a predominant population of cleavage products that correspond to cleavage at the base of the flap (39 bp) (, lane 2). A fully expanded product was also observed upon incubation with Cdc9 (75 bp) (, lane 3). The combined addition of Rad27 and Cdc9 produced both a correctly processed (66 bp) and fully expanded product (, lane 4). Upon titration of Msh2-Msh3, however, smaller expansion products (69 and 72 bp) were predominant over the larger fully expanded product (, lanes 5–9). These products are not observed with substrates containing short equilibrating flaps consisting of randomly generated sequences (data not shown). Specifically, Msh2-Msh3 causes up to a ~3.3 fold increase in the accumulation of smaller expansion products (, lane 9). These data are all consistent with Msh2-Msh3 binding and stabilizing small TNR structures that form within the flaps.